Cytotoxic effects of extracts obtained from plants of the Oleaceae family: bio-guided isolation and molecular docking of new secoiridoids from Jasminum humile

Abstract Context Traditionally, Oleaceae plants are used to treat many diseases, such as rheumatism, hypercholesterolaemia, or ulcers. Objectives To investigate the cytotoxic potential of Jasminum humile L., Jasminum grandiflorum L., and Olea europaea L. (Oleaceae) extracts against selected human cancer cells lines, followed by a phytochemical investigation of the most potent one. Materials and methods The 95% ethanol extracts of aerial parts of three oleaceous plants were examined for their cytotoxicity against HepG-2, MCF-7, and THP-1 cell lines using MTT assay and doxorubicin (positive control). J. humile was bio-selected and submitted to bio-guided fractionation. Chromatographic workup of ethyl acetate and n-butanol fractions afforded two new compounds; 1-methoxyjasmigenin (1) and 1-methyl-9-aldojasmigenin (2), along with five known ones (3–7). Structures were unambiguously elucidated using 1D/2D NMR and ESI-HRMS. Isolated compounds were assessed for their anti-proliferative potential, and both selectivity index and statistical significance were determined. Molecular docking was conducted against the Mcl-1 receptor using (AZD5991) as a standard. Results Jasmoside (5) was the most potent anticancer compound showing IC50 values of 66.47, 41.32, and 27.59 µg/mL against HepG-2, MCF-7, and THP-1 cell lines, respectively. Moreover, isojasminin (4) exhibited IC50 values of 33.49, 43.12, and 51.07 µg/mL against the same cell lines, respectively. Interestingly, 5 exhibited the highest selectivity index towards MCF-7 and THP-1, even greater than doxorubicin. Molecular docking results were in full agreement with the MTT assay and the proposed SAR. Conclusion In this study, two new compounds were purified. The biological activity highlighted jasmoside (5) as a lead anticancer drug for further future investigation.


Introduction
During the past centuries, our ancestors considered plants as effective medicinal remedies. Currently, many people all over the world still use plants, as an alternative and/or traditional medicine. In Africa, more than 80% of the population depends on herbal products and traditional medicine for healthcare during their lifetime. Even in developed countries, herbal products are used under the term 'complimentary' medicine (Akinyemi et al. 2018). The majority of these plants are still under investigation. Phytochemical and biological investigations could maximize the therapeutic effects, and alleviate the potential side effects of herbal drugs. Moreover, this may result in the discovery of novel compounds, which could contribute to finding new mechanisms and activities.
Oleaceae (the olive family) includes 28 genera and about 900 species (Akhtar et al. 2021). It is well-known for its multiple nutritional, perfumery, and horticultural uses. Moreover, oleaceous plants are widely used as traditional treatments. In Belarus, the buds of Syringa vulgaris L. are processed into wine and used for the treatment of joint pain. In Southern Italy, the bark of Fraxinus ornus L. is often used as antidiarrheal and hypocholesterolemic. The fruits of the Chinese tree Ligustrum lucidum W.T. Aiton are used to improve both liver and kidney functions. In Greece, the leaves of Olea europaea L. are used as hypotensive. The volatile oil extracted from O. europaea is used as an antirheumatic and used as a laxative in Oman. In addition, olive oil has been reported to have both anticancer and antioxidant effects (Huang et al. 2019;El Haouari et al. 2020;De Bruno et al. 2021).
Jasmines (Jasminum spp.) are widely cultivated flowering plants of the Oleaceae family. In addition to their pleasant fragrances, different species of this genus were proven to have various biological activities. Jasminum sambac L. has traditionally been used as an analgesic and antiseptic in addition to its use as a fragrance in skin care products. Its essential oil was reported to have antimicrobial and antioxidant effects (Abdoul-Latif et al. 2010). Jasminum grandiflorum is used in folk medicine as an antiulcer (Venkateswararao and Venkataramana 2013). Leaves are used in odontalgia, leprosy, skin diseases, ottorrhoea, otalgia, strangury, and dysmenorrhoea (Sandeep et al. 2009). The aerial parts of J. grandiflorum were reported to have anti-anthelmintic activity (Hussein et al. 2021). Jasminum humile L. is used traditionally as an astringent, cardiac tonic, and for treating hard lumps and chronic fistulas . In India, it is used as a tonic and also as a cure for ringworms (Singh et al. 2021). Several oleaceous plants, such as O. europaea (Zaïri et al. 2020;Essafi Rhouma et al. 2021) and other Jasminum species (Jantova et al. 2001;Hue Ngan et al. 2008;Kalaiselvi et al. 2011;Wei et al. 2021) was reported to have potent cytotoxic and anticancer activities. Unfortunately, few researchers have investigated the biological activities of J. humile, and little is reported regarding its phytochemical constituents.
In 2020, breast cancer was found to be the most commonly diagnosed cancer, while liver cancer ranked third on the list of cancer-related deaths (Sung et al. 2021). Both types are pervasive in Egypt, and they are both the first and second leading causes of cancer cases (WHO 2020). Moreover, leukaemia accounts for $35.6% of all cancer cases among Egyptian children (Alburaiki et al. 2021). In this study, the cytotoxic effects of three Oleaceous plants (J. humile L., J. grandiflorum L., and O. europaea L.) were investigated using a MTT assay against two solid tumours (liver cancer and breast cancer), as well as leukaemia (a liquid tumor). The bioguided selection was conducted and J. humile was selected and subjected to successive fractionation. Chromatographic workup of the respective bioactive fractions (ethyl acetate and n-butanol) yielded two new secoiridoids (1-2) and five known compounds (3-7). The isolated compounds were investigated for their cytotoxic activity, and selectivity index, as well as statistical significance, were determined (compared to the reference drug doxorubicin).
A molecular docking study of the seven isolated compounds was conducted against Mcl-1, a protein member of the Bcl-2 family that inhibits apoptosis in many cancerous cells and consequently keeps their survival. Elevated expression of Mcl-1 levels was recorded in many cases of tumorigenesis and/or resistance to anti-tumor agents as well. Therefore, many efforts have been directed towards the development of potent cytotoxic agents targeting the Mcl-1 receptor (Eliaa et al. 2020;Samra et al. 2021). Herein, the structure characterization, cytotoxic activity, statistical significance, molecular docking, and a proposed structure-activity relationship of the isolated constituents were investigated.

Plant materials
The aerial parts of J. humile, J. grandiflorum, and O. europaea were collected in November 2018 from the botanical garden of Mansoura University, Egypt. Plants were verified by Dr. Ibrahim Mashaly, Professor of Ecology, Faculty of Science, Mansoura University, Egypt. Freshly collected plant materials were dried in the shade at room temperature, powdered and kept at 4 C for further investigation. The voucher specimens were deposited in the herbarium of Pharmacognosy Department, Faculty of Pharmacy, Mansoura University, and given the code (Jh-01) for J. humile, (Jg-03) for J. grandiflorum, and (Oe-07) for O. europaea.

Extraction and fractionation
The air-dried powdered materials of J. humile (1300 g), J. grandiflorum (620 g), and O. europaea (580 g) were extracted with 95% ethanol (Merck, Germany) by cold maceration till exhaustion. The solvent was then removed by vacuum distillation at a temperature of no more than 40 C using a rotary evaporator (Heidolph, Laborota 4000), and the solvent-free dried extracts were weighed to determine the percentage yields (13.6, 12.1, and 14.7% w/w, respectively). The dried extracts underwent biological investigations and a bio-guided selection was performed with J. humile as the most active. The ethanol extract of J. humile (95 g) was selected suspended in water and subjected to successive liquid-liquid fractionation using solvents of increasing polarities viz., petroleum ether, methylene chloride, ethyl acetate, and water-saturated n-butanol (Merck, Germany). In each case, the solvent was removed by vacuum distillation at a temperature not exceeding 40 C using a rotary evaporator. A bio-guided selection was performed again where both the ethyl acetate and n-butanol fractions were chosen. The solvent-free extract residues were then weighed and preserved for the process of isolating their active compounds.
Apparatus, equipment, and general techniques 1 H and 13 C-NMR spectra were obtained utilizing a Bruker DRX 400 NMR spectrometer (Bruker Daltonics Inc., MU, Egypt). Chemical shifts (d) were expressed in ppm with reference to the TMS resonance. ESI-HRMS was determined using LCMS-IT-TOF (Shimadzu, Tokyo, Japan). The MS instrument was operated using an ESI source in both positive and negative ionization modes with survey scans acquired from m/z 100-2000 for MS and m/z 50-1500 for MS/MS. The ionization parameters were as follows: probe voltage, ± 4.5 kV; nebulizer gas flow, 1.5 L/min; CDL temperature, 200 C; heat block temperature, 200 C. UV spectra were obtained using a UV-visible spectrophotometer (Shimadzu 1601 PC, model TCC240, Kyoto, Japan). Infra-red (IR) spectra were obtained by using an FTIR spectrometer 620, Jasco (Tokyo, Japan). Optical rotations were measured with a Jasco DIP-370 polarimeter.

Calculation of the selectivity index (SI)
The selectivity index (SI) was obtained after dividing the IC 50 value of Vero cells (which can be expressed as CC 50 ) by the specific IC 50 of cancer cell lines. High (SI) indicates high anticancer activity and low cellular toxicity. Selectivity was considered to be valuable for those with (SI) greater than three (Oliveira et al. 2015;Tsemeugne et al. 2021;Yousefbeyk et al. 2022).  Table 1.

Spectroscopic data of the compounds
(-)-1-Methyl-9-aldojasmigenin (2) The spectroscopic data of the previous compounds are reported in the Supplemental Material.

Statistical analysis
All statistical analyses were performed using GraphPad Prism version 9.2.0 to calculate the half-maximal inhibitory concentration (IC 50 ) and the half-maximal cytotoxic concentration (CC 50 ) where the level of significance was set at (p > 0.05). Quantitative data were expressed as mean ± standard deviation (SD). GraphPad Prism version 9.2.0 was also used to create multiple bar charts of the cytotoxic activity and cell viability.

Molecular docking study
Validation of the docking process using the MOE program At first, a program validation process was carried out to confirm the validity of the MOE program (Inc. 2019). Therefore, a redocking process of the co-crystallized inhibitor (AZD5991) was performed within its binding pocket. The program validity was concluded by observing a similar binding mode of the re-docked AZD5991 (green) relative to its native co-crystallized one (red), besides, a low RMSD value (1.89). Both the 2D and 3D pictures describing the superimposition of the redocked co-crystallized inhibitor AZD5991 (green) over its native one (red) are depicted in the Supplemental Material.
Docking studies using Molecular Operating Environment 2019.012 suite (MOE 2019) have been applied to identify the mechanism of action for the seven isolates (1-7) of J. humile as Mcl-1 inhibitors using molecular docking studies. This was performed based on their binding scores and interactions as well. Meanwhile, AZD5991 was utilized as a reference standard.
Identified isolates from J. humile and AZD5991 preparation The ChemDraw Professional program was used to sketch the 2D chemical structures of the isolated compounds (1-7) and the standard AZD5991 (8). Each molecule was prepared separately by its introduction into the MOE window where it was transformed to the 3D form, the partial charges were calculated, and the energy was reduced to be ready for the docking process as previously described (Abo Elmaaty et al. 2021). Moreover, all prepared compounds were inserted into a single database and saved as an extension of (.mdb) to be used during the docking step.

Mcl-1 protein preparation
The Mcl-1 protein was downloaded from the Protein Data Bank (PDB code: 6FS1) (Bowman et al. 2006). It was prepared for docking by patching, adding hydrogen bonds, and reducing energy as previously described in detail El-Shershaby et al. 2021).
Docking the prepared database (1-8) into the Mcl-1 binding pocket A general docking process was applied using the aforementioned database of the ligand and the site finder approach was used to determine the docking site inside the protein receptor (Mahmoud et al. 2021). Program specifications were adjusted as previously demonstrated Soltan et al. 2021). Besides, one mode of each tested compound was selected according to their binding scores and RMSD refine values.
Further investigation of the NMR spectra revealed the absence of an anomeric sugar proton signal observed at d H 4.81 in 3 and the appearance of an aliphatic methoxy signal at d H/C 3.38/55.9 in 1. The key HMBC correlations (Figure 2) from OCH 3 -(d H 3.38) to C-1 (d C 102.7) secured the position of this methoxy group at C-1, which confirmed that compound 1 is the methoxy derivative of jasminin where an aliphatic methoxy group replaces the sugar moiety linked to C-1. Careful inspection of the ROESY spectrum of 1 revealed that the allocation of substituents at chiral centres was entirely consistent with those published for jasminin (3), as depicted in Figure 1. This was further confirmed by the same sign of the optical rotation of (1) [a] 20 D À89.5 (MeOH) and the sign of co-isolated derivative jasminin (3) [a] 20 D À110.3 (MeOH) when measured under the same conditions. Therefore, 1 was elucidated as a new jasminin congener, isolated for the first time from a natural source, and given the trivial name 1-methoxyjasmigenin.

Structure elucidation of compound 2
Compound (2) was isolated from the ethyl acetate fraction as a white amorphous powder (3.5 mg), soluble in both MeOH and ethyl acetate and demonstrating UV (MeOH) absorbance maximum at 238 nm, [a] 20 D À171.1 (c 0.023, MeOH) and IR bands (KBr) at 3441, 1637, and 1015 cm À1 indicative for iridoids (Inoue et al. 1985(Inoue et al. , 1991Chang et al. 2020). Thin layer chromatography using the solvent system Pet. Ether-EtOAc (40:60 v/v) revealed a spot with R F value of 0.51, quenched UV light at 254 nm, which acquired a brownish colour after heating with vanillin/sulfuric acid spray reagent at 105 C for 1 min. It exhibited an ion peak at m/z 379.1758 [M À H] À (calcd, 379.1763) in the ESI-HRMS, which is comparable with the molecular formula C 20 H 28 O 7 and indicates a DBE of seven.
The NMR spectral features of 2 (Table 1) were quite comparable to those of 1, indicating the presence of 7,8-seco-cyclopenta[c]-pyranoid skeleton for secoiridoids (Figure 1). The disappearance of the methoxy group signals at d H/C 3.38/55.9 in 1 and the presence of an extra aliphatic methyl resonating at d H/C 1.52/17.7 (H-1 0 , d, J ¼ 6.7 Hz) in 2, along with the up-field shift of the methine group to d H/C 4.38/69.9 (H-1, qd, J ¼ 6.6, 2.5) in 2 compared to that at d H/C 102.7/5.31 (H-1, s) in 1, indicates the replacement of the methoxy group in 1 by the methyl group in 2. Moreover, the characteristic vinyl methyl doublet detected at d H 1.73 in 1 disappeared and an aldehydic group at d H/C 9.64/201.6 (H-8, d, J ¼ 2.5 Hz), as well as an extra methine signal at d H/C 2.73/51.1 (H-9, m), were observed in 2 instead. These deductions were confirmed by the aforementioned coupling constants, ( 1 H-1 H) COSY and HMBC correlations ( Figure  2). The assignment of the aldehyde group at C-9 was supported by HMBC correlations from H-1 (d H 4.38) to C-8 (d C 201.6), from H-9 (d H 2.73) to C-8 and C-5 (d C 27.9) and from H-8 (d H 9.64) to C-5. In addition, the HMBC correlations from CH 3 -1 0 (d H 1.52) to C-1 (d C 69.9) and C-9 secured its position to C-1.
It is noteworthy that compound 2 has an extra chiral centre at C-9 that is not present in compounds 1 and 3. The relative configuration at C-9 was established by analysis of the coupling constants and the ROESY experiment. The small coupling constant between H-1 and H-9 (J ¼ 2.5 Hz) confirmed their gauche or cis relationship. This was confirmed unambiguously by the cross peak in the ROESY spectrum between CH 3 -1 0 (d H 1.52) and the aldehydic proton H-8 at (d H 9.64), confirming that they are co-faced in the molecule as depicted in Figure 2. The stereochemistry of the iridane moiety was deduced from the ROESY spectrum. It was found to have the same configuration as in 1 and jasminin (3).
According to all the previous data, compound 2 was designated as 1-methyl-9-aldojasmigenin, a new compound isolated for the first time from a natural source.

Cytotoxic concentrations of tested samples
The CC 50 for all examined samples was assessed on VERO cells using an MTT assay ( Table 2). The CC 50 of the ethanolic extracts J. humile, J. grandiflorum, and O. europaea varied from 63.39 to 108.60 mg/mL. The petroleum ether, methylene chloride, ethyl acetate, and n-butanol fractions of J. humile showed CC 50 values ranging from 26.51 to 108.13 mg/mL, while the isolated compounds exhibited cytotoxicity range from 44.73 to 210.17 mg/mL.

Cytotoxic activity of tested samples
All samples were evaluated for their cytotoxicity against THP-1, HepG-2, and MCF-7 cell lines in a concentration-dependent manner, and the results are shown in Table 2. The ethanolic extract of J. humile demonstrated the lowest IC 50 ; i.e., the highest cytotoxic activity against both THP-1 (46.63 lg/mL) and HepG-2 (59.47 lg/mL), as well as very close (IC 50 ) to that of O. europaea against MCF-7 cell line (47.49 vs. 47.22 mg lg/mL). Based on these experimental results, the ethanolic extract of the aerial parts of J. humile was selected for further bio-guided fractionation using different organic solvents.
The ethyl acetate and n-butanol fractions significantly inhibited cell growth of the breast cancer cell line; MCF-7 after using different concentrations (compared to the reference drug doxorubicin) ( Table 2). According to the guidelines of the US National Cancer Institute, the crude extract may be considered as highly active for an IC 50 30 mg lg/mL (Aoussar et al. 2020). Therefore, the ethyl acetate (IC 50 ¼ 22.78 mg/mL) and n-butanol (IC 50 ¼ 26.59 mg/mL) fractions could be considered as highly active against the MCF-7 cell line, and the results were statistically significant compared to doxorubicin. The ethyl acetate fraction showed the highest cytotoxic activity against all cell lines, with an IC 50 of 30.08, 22.78, and 36.86 mg/mL against HepG-2, MCF-7, and THP-1 cell lines, respectively. The n-butanol fraction also demonstrated high cytotoxic activity, with IC 50 of 70.28, 26.59, and 57.37 mg/mL against HepG-2, MCF-7, and THP-1 cell lines, respectively, and the two fractions were selected for chromatographic isolation of their active components.
The activity-guided chromatographic isolation led to identifying five compounds (1-4 and 6) from the ethyl acetate fraction and two compounds from the n-butanol fraction (5 and 7). All isolated compounds were investigated for their cytotoxic activity (Table 2). Different concentrations were used and statistical significance was determined compared to the reference drug doxorubicin. Compound (5) showed the highest effect against THP-1 cell line. It is evident that it is very promising for continued research as an anticancer agent due to its high potency (66.47 lg/mL against HepG-2, IC 50 of 41.32 lg/mL against MCF-7, and 27.59 lg/mL against THP-1) as well as high selectivity (SI ¼ 3.16 for HepG-2, 5.09 for MCF-7, and 7.62 for THP-1) on the tested cell lines (Table 2).

Docking studies
The molecular docking study of the seven identified isolates from J. humile (1-7), besides docking AZD5991, as a reference standard, was carried out against the binding pocket of the Mcl-1 receptor. The binding scores were recorded in the following order; docked AZD5991 (8) > jasmoside (5) > isojasminin (4) > jasminin (3) > 1-methyl-9-aldojasmigenin (2) > 1-methoxyjasmigenin (1) > coumarin (6) > D-mannitol (7). Notably, AZD5991 inhibitor (8) was found to form both an ionic bond and a hydrogen bond with Arg263 contrary to the other surrounding amino acids, which was recommended as the most important one for the Mcl-1 receptor inhibition effect. The binding scores and interactions within the pocket amino acids of the Mcl-1 receptor are depicted in Table 3.
From (Table 3), we can conclude that (3-5) isolates showed the best scores (À6.97, À7.34, and À8.42 kcal/mol, respectively) compared to that of the docked AZD5991 (8) (À8.60 kcal/mol). This recommends the proposed mechanism of action for isolates identified as promising inhibitors of the Mcl-1 receptor. Compound (3) was proven to form two H-bonds with Arg263 amino acid at 2.92 and 3.12 Å. In contrast, compound (4) was stabilized inside the binding pocket through the formation of one H-bond with Arg263 amino acid at 3.12 Å and one H-pi bond with His224 at 4.41 Å. However, compound (5) made three H-bonds with Lys214, Arg263, and Met231 at 3.03, 3.11, and 3.81 Å, respectively (Table 4). Finally, the docked AZD5991 inhibitor (8) indicated the formation of one H-bond and two ionic bonds with Arg263 amino acid at 3.04, 3.08, and 3.19 Å, respectively. Also, it showed the formation of two pi-H bonds with Met250 and Gly230 amino acids at 3.97 and 4.61 Å, respectively.
Based on the above, we can conclude that the isolates (3-5) demonstrated better binding scores towards the binding pocket of Mcl-1 protein despite the formation of a small number of interactions, indicating their elevated affinity for the tested receptor pocket. This largely demonstrates their expected intrinsic activity as Mcl-1 inhibitors as well.

Structure-activity relationship study
With respect to the SAR-based on both the aforementioned in vitro and in silico results for compounds (1-7) isolated from J. humile (Figure 1), we can conclude that: (a) Compound (5) (the dimer secoiridoid) achieved the best cytotoxic activities towards the examined cell lines which can be attributed to the presence of two glucose units at the two dihydro-2H-pyran rings that can improve its fitting within the active site of the Mcl-1 receptor. (b) In contrast, compound (4) showed slightly better antitumor activities than compound (3) which may be due to the presence of a single glucose moiety at the dihydro-2H-pyran ring in both cases. (c) Based on the above, we can conclude that the presence of sugar moieties (glucose sugar) significantly improves the cytotoxic activities of secoiridoids in targeting Mcl-1 receptor as a proposed mechanism of action. (d) Moreover, the presence of a methyl group on the dihydro-2H-pyran ring of compound (2) improved the cytotoxic activities compared to the methoxy substituent of the compound (1). (e) Additionally, the presence of the aldehydic group at position 9 of compound (2) may contribute to the enhanced antitumor activities. (f) Finally, the lower cytotoxic activities of compounds (6) and (7) were sequential.

Conclusions
In the present study, the aerial parts of J. humile, J. grandiflorum, and O. europaea were examined to determine their cytotoxic activity against HepG-2, MCF-7, and THP-1. J. humile was the most potent and thus was subjected to successive fractionation. The fractions were tested on the same cell lines and two fractions (ethyl acetate and n-butanol fractions) were selected for further chromatography. This bioassay-guided chromatographic isolation from J. humile yielded 7 compounds, namely [1methoxyjasmigenin (1), 1-methyl-9-aldojasmigenin (2), jasminin (3), isojasminin (4), jasmoside (5), coumarin (6), and D-mannitol (7)]; two of them, (1) and (2), have been designated as new compounds. All isolates were evaluated for their cytotoxic activities using the MTT assay and molecular docking studies. Statistical significance was determined in comparison to the reference drug doxorubicin and the selectivity index was determined.
The study introduces compound (5) as a valuable lead compound demonstrating in vitro cytotoxic activity as well as reasonable selectivity towards HepG-2, MCF-7, and THP-1 cell lines, while compound (4) showed a promising potency and selectivity towards HepG-2 cells. Compound (3) and the new secoiridoids; (1) and (2) also showed marked cytotoxic activities. All isolates were examined against Mcl-1 using molecular docking studies. A significant match was found between the computational docking and the in vitro studies. Their IC 50 and docking scores recommended them, especially the isolated secoiridoids; (1-5), as very promising cytotoxic drug candidates since they demonstrated better binding scores towards the binding pocket of Mcl-1 protein despite inducing limited interactions; indicating a great affinity for the tested receptor pocket. This strongly suggests their expected intrinsic activity as Mcl-1 inhibitors as well. A proposed SAR was introduced for further semi-synthesis and/or synthesis of effective future cytotoxic drugs. Further biological investigations and in vivo studies are recommended to evaluate their potential as cytotoxic therapeutics and/or semi-synthetic precursors.

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Funding
The author(s) reported there is no funding associated with the work featured in this article.